Background: Lipopolysaccharide (LPS), the major component of the outer membrane of gram-negative bacteria, can activate immune cells including macrophages. Activation of macrophages in the central nervous system (CNS) contributes to neuronal injury. Bowman-Birk inhibitor (BBI), a soybean-derived protease inhibitor, has anti-inflammatory properties. In this study, we examined whether BBI has the ability to inhibit LPS-mediated macrophage activation, reducing the release of pro-inflammatory cytokines and subsequent neurotoxicity in primary cortical neural cultures.

Methods: Mixed cortical neural cultures from rat were used as target cells for testing neurotoxicity induced by LPS-treated macrophage supernatant. Neuronal survival was measured using a cell-based ELISA method for expression of the neuronal marker MAP-2. Intracellular reactive oxygen species (ROS) production in macrophages was measured via 2', 7'-dichlorofluorescin diacetate (DCFH2DA) oxidation. Cytokine expression was determined by quantitative real-time PCR.

Results: LPS treatment of macrophages induced expression of proinflammatory cytokines (IL-1β, IL-6 and TNF-α) and of ROS. In contrast, BBI pretreatment (1-100 μg/ml) of macrophages significantly inhibited LPS-mediated induction of these cytokines and ROS. Further, supernatant from BBI-pretreated and LPS-activated macrophage cultures was found to be less cytotoxic to neurons than that from non-BBI-pretreated and LPS-activated macrophage cultures. BBI, when directly added to the neuronal cultures (1-100 μg/ml), had no protective effect on neurons with or without LPS-activated macrophage supernatant treatment. In addition, BBI (100 μg/ml) had no effect on N-methyl-D-aspartic acid (NMDA)-mediated neurotoxicity.

Conclusions: These findings demonstrate that BBI, through its anti-inflammatory properties, protects neurons from neurotoxicity mediated by activated macrophages.

Figure 3: Impact of BBI on rat cortical neuron cultures. (A) Effect of BBI on LPS/MΦ-mediated neurotoxicity. Supernatant either from LPS- (100 ng/ml) activated macrophage cultures (LPS/MΦ SN) or from BBI- (1-100 μg/ml) treated and LPS-activated macrophage culture (BBI/LPS/MΦ SN) was used to treat rat cortical neurons (1:10). Untreated and unactivated macrophage supernatant was used as a negative control (MΦ SN). Data are expressed as mean ± SD for three independent experiments. (*P < 0.05, **P < 0.01, BBI/LPS/MΦ SN vs LPS/MΦ SN without BBI). (B) Effect of BBI on neuron death for rat cortical neurons. Rat cortical neurons were directly treated with BBI at the indicated concentrations or with supernatant from either unactivated and untreated macrophage culture (MΦ SN) or BBI- (1-100 μg/ml) treated macrophage culture (BBI/MΦ SN) for 24 h. Data are expressed as mean ± SD for three independent experiments. (C) Effect of BBI treatment on neurotoxicity of LPS-activated macrophages. Cortical neuronal cultures were treated with LPS-activated macrophage supernatant in the presence or absence of BBI for 24 h. Data are expressed as mean ± SD for three independent experiments.

Mentions:
We first examined whether supernatant from LPS-activated macrophage cultures could induce neuron death. Although LPS, when directly added to the rat cortical neuron cultures, had no cytotoxic effect (Figure 1A), supernatant from LPS-activated macrophage cultures induced the neuron death, which was evidenced by decreased MAP-2 expression (Figure 1A and 1B). This LPS/macrophage supernatant-mediated neuronal death was positively related to amount of supernatant added to the rat cortical neuron cultures (Figure 1A). In addition, the concentration of LPS used for macrophage activation was positively associated with degree of neurotoxicity of the LPS/macrophage supernatants (Figure 1B). In contrast, supernatant from BBI-pretreated and then LPS-activated macrophage cultures produced reduced neurotoxicity, compared to that from non-BBI-pretreated cultures (Figure 2 and Figure 3A). Immunofluorescence assays also demonstrated that BBI pre-treatment of macrophages could alleviate the neurotoxicity of LPS-activated macrophages (Figure 2). The direct addition of supernatant from BBI-treated macrophage cultures or of BBI to the neuronal cultures had no cytotoxic effect (Figure 3B). In addition, BBI treatment of neuronal cells had no protective effect against the neurotoxicity of supernatant from LPS-activated macrophage cultures (Figure 3C).

Figure 3: Impact of BBI on rat cortical neuron cultures. (A) Effect of BBI on LPS/MΦ-mediated neurotoxicity. Supernatant either from LPS- (100 ng/ml) activated macrophage cultures (LPS/MΦ SN) or from BBI- (1-100 μg/ml) treated and LPS-activated macrophage culture (BBI/LPS/MΦ SN) was used to treat rat cortical neurons (1:10). Untreated and unactivated macrophage supernatant was used as a negative control (MΦ SN). Data are expressed as mean ± SD for three independent experiments. (*P < 0.05, **P < 0.01, BBI/LPS/MΦ SN vs LPS/MΦ SN without BBI). (B) Effect of BBI on neuron death for rat cortical neurons. Rat cortical neurons were directly treated with BBI at the indicated concentrations or with supernatant from either unactivated and untreated macrophage culture (MΦ SN) or BBI- (1-100 μg/ml) treated macrophage culture (BBI/MΦ SN) for 24 h. Data are expressed as mean ± SD for three independent experiments. (C) Effect of BBI treatment on neurotoxicity of LPS-activated macrophages. Cortical neuronal cultures were treated with LPS-activated macrophage supernatant in the presence or absence of BBI for 24 h. Data are expressed as mean ± SD for three independent experiments.

Mentions:
We first examined whether supernatant from LPS-activated macrophage cultures could induce neuron death. Although LPS, when directly added to the rat cortical neuron cultures, had no cytotoxic effect (Figure 1A), supernatant from LPS-activated macrophage cultures induced the neuron death, which was evidenced by decreased MAP-2 expression (Figure 1A and 1B). This LPS/macrophage supernatant-mediated neuronal death was positively related to amount of supernatant added to the rat cortical neuron cultures (Figure 1A). In addition, the concentration of LPS used for macrophage activation was positively associated with degree of neurotoxicity of the LPS/macrophage supernatants (Figure 1B). In contrast, supernatant from BBI-pretreated and then LPS-activated macrophage cultures produced reduced neurotoxicity, compared to that from non-BBI-pretreated cultures (Figure 2 and Figure 3A). Immunofluorescence assays also demonstrated that BBI pre-treatment of macrophages could alleviate the neurotoxicity of LPS-activated macrophages (Figure 2). The direct addition of supernatant from BBI-treated macrophage cultures or of BBI to the neuronal cultures had no cytotoxic effect (Figure 3B). In addition, BBI treatment of neuronal cells had no protective effect against the neurotoxicity of supernatant from LPS-activated macrophage cultures (Figure 3C).

Background: Lipopolysaccharide (LPS), the major component of the outer membrane of gram-negative bacteria, can activate immune cells including macrophages. Activation of macrophages in the central nervous system (CNS) contributes to neuronal injury. Bowman-Birk inhibitor (BBI), a soybean-derived protease inhibitor, has anti-inflammatory properties. In this study, we examined whether BBI has the ability to inhibit LPS-mediated macrophage activation, reducing the release of pro-inflammatory cytokines and subsequent neurotoxicity in primary cortical neural cultures.

Methods: Mixed cortical neural cultures from rat were used as target cells for testing neurotoxicity induced by LPS-treated macrophage supernatant. Neuronal survival was measured using a cell-based ELISA method for expression of the neuronal marker MAP-2. Intracellular reactive oxygen species (ROS) production in macrophages was measured via 2', 7'-dichlorofluorescin diacetate (DCFH2DA) oxidation. Cytokine expression was determined by quantitative real-time PCR.

Results: LPS treatment of macrophages induced expression of proinflammatory cytokines (IL-1β, IL-6 and TNF-α) and of ROS. In contrast, BBI pretreatment (1-100 μg/ml) of macrophages significantly inhibited LPS-mediated induction of these cytokines and ROS. Further, supernatant from BBI-pretreated and LPS-activated macrophage cultures was found to be less cytotoxic to neurons than that from non-BBI-pretreated and LPS-activated macrophage cultures. BBI, when directly added to the neuronal cultures (1-100 μg/ml), had no protective effect on neurons with or without LPS-activated macrophage supernatant treatment. In addition, BBI (100 μg/ml) had no effect on N-methyl-D-aspartic acid (NMDA)-mediated neurotoxicity.

Conclusions: These findings demonstrate that BBI, through its anti-inflammatory properties, protects neurons from neurotoxicity mediated by activated macrophages.